Pantothenic acid is produced all over the world on a scale of several thousand tonnes per year. A large part of the pantothenic acid produced is used for feeding economically useful animals such as poultry and pigs. Demand is increasing.
Pantothenic acid may be prepared by chemical synthesis or biotechnically by the fermentation of suitable microorganisms in suitable nutrient solutions. In the case of chemical synthesis, DL-pantolactone is an important precursor. It is prepared in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide. In further process steps, the racemic mixture is separated and D-pantolactone is condensed with .beta.-alanine, thus producing D-pantothenic acid.
The typical commercial form is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture of D,L-pantothenic acid can also be used.
The advantage of fermentative preparation using microorganisms is the direct formation of the desired stereoisomeric form, that is the D-form, which contains no L-pantothenic acid.
Various types of bacteria such as, for example, Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes and also yeasts such as, for example, Debaromyces castellii may, as shown in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, produce D-pantothenic acid under suitable conditions of fermentation. Particularly suitable microorganisms are the derivatives of Escherichia coli IF03547 described there, such as e.g. the strains FV5069/pFV31 or FV5069/pFV202.
During the fermentative preparation of D-pantothenic acid, as described in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, a microorganism capable of D-pantothenic acid production is cultivated in a suitable nutrient and the D-pantothenic acid produced is then isolated in a costly manner, purified and prepared as the calcium salt.
Suitable nutrient media contain a source of carbon such as e.g. glucose or starch flour hydrolysate or sucrose or molasses, precursors such as e.g. .beta.-alanine, D,L-pantoic acid or D,L-pantolactone, a source of nitrogen such as e.g. ammonium sulfate, a source of phosphorus such as e.g. potassium phosphate and other salts, trace elements and vitamins and optionally complex media additives such as e.g. yeast extract. The microorganisms are then incubated in this medium at a suitable pH, with appropriate aeration and stirring, wherein these then excrete D-pantothenic acid.
According to the current prior art, which is represented by WO96/33283 and EP-A-0 590857, the calcium salt of D-pantothenic acid is obtained from the pantothenic acid-containing fermentation broth by costly isolation and purification. After initial isolation of the biomass by filtering or centrifuging, further processing of the filtrate is performed by purification using active carbon or column chromatography. After reaction of the solutions obtained in this way with calcium hydroxide, the desired Ca salt crystallises out.
According to WO 96/33283 the filtrate is decoloured with active carbon in the first column. The pH is adjusted to 3.0 with concentrated hydrochloric acid and the liquid is then continuously purified over two further columns packed with active carbon. Elution of the D-pantothenic acid is achieved with the aid of methyl alcohol. After the subsequent neutralisation step using Ca(OH).sub.2 powder, a solution is obtained from which calcium D-pantothenate is recovered by crystallisation at 5.degree. C.
In the method described in EP-A-0 590 857, the filtrate is first purified with the aid of cation and anion exchanger columns. Elution is performed with hydrochloric acid. The eluted fraction is then neutralised with Ca(OH).sub.2, active carbon is added thereto and the mixture is filtered. The filtrate obtained is then extracted into a low molecular weight alcohol (methanol, ethanol, isopropanol)and calcium D-pantothenate is obtained by crystallisation.
The calcium D-pantothenate prepared in the way described above is used as an additive in feedstuffs for animal nutrition.